Characterization of the human factor VIII procoagulant protein with a heterologous precipitating antibody.
The human factor VIII procoagulant protein (VIIIC) was purified from the VIIIC--factor VIII--related antigen (VIIIRAg) complex in commercial factor VIII concentrate by immunoadsorbent chromatography with a monoclonal anti-VIIIRAg antibody bound to Sepharose. In this complex, VIIIC is noncovalently bound to factor VIII-related antigen. The VIIIC eluted from the complex was free of VIIIRAg as determined by immunoassay and had a specific activity of 2,294 VIIIC units/mg of protein, representing a 164,000-fold purification from plasma. Electrophoresis of this VIIIC preparation in reduced sodium dodecyl sulfate containing 5% polyacrylamide slab gels and subsequent staining with Coomassie blue showed the VIIIC to be a strongly staining doublet of Mrs 79,000 and 80,000 and more faintly staining bands of up to Mr 188,000. Treatment of the VIIIC with catalytic amounts of thrombin resulted in diminution or complete disappearance of all of these bands and appearance of a doublet of Mrs 71,000 and 72,000, a band at Mr 54,000, and material of lower Mr. The purified VIIIC was used to produce a precipitating heterologous anti-VIIIC antibody, which was shown to be monospecific for VIIIC after adsorption with factor VIII-depleted commercial concentrate. Use of this antibody in crossed immunoelectrophoresis positively identified the VIIIC bands in the sodium dodecyl sulfate/polyacrylamide gels. These techniques have allowed the identification of VIIIC protein and description of its extensive Mr heterogeneity.
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D005169:Factor VIII / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D011498:Protein Precursors / Q000276:immunology; D013917:Thrombin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011498:Protein Precursors; D005169:Factor VIII; D013917:Thrombin